Electrode compositions and configurations for electrochemical bioreactor systems

ABSTRACT

Electrodes and configurations for electrochemical bioreactor systems that can use electrical energy as a source of reducing power in fermentation or enzymatic reactions and that can use electron mediators and a biocatalyst, such as cells or enzymes, to produce electricity are disclosed. Example electrodes in the system may comprise: (1) neutral red covalently bound to graphite felt (FIG.  1 ); (2) a carboxylated cellulose bound to the graphite fell, neutral red bound to the carboxylated cellulose, NAD +  bound to the graphite fell, and an oxidoreductase (e.g., fumarate reductase) bound to the graphite fell; or (3) a metal ion electron mediator bound to graphite. Various biocatalysts, such as an oxidoreductase, cells of  Actinobacillus succinogenes,  cells of  Escherichia coli,  and sewage sludge, are suitable for use in the electrochemical bioreactor system.

CROSS-REFERENCES To RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/294,943 filed May 31,2001, U.S. Provisional PatentApplication No. 60/338,245 filed Nov. 8, 2001 and U.S. ProvisionalPatent Application No. 60/353,037 filed Jan. 30, 2002.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] Not Applicable.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates to electrochemical bioreactor systems thatcan provide electrical enhancement of chemical synthesis and/orfermentations and/or biotransformations when supplied with electricity,that can generate electrical current detectable at a load, and that mayused in chemical or biochemical sensing devices. In particular, theinvention relates to improved electrodes for electrochemical bioreactorsystems that can use electrical energy as a source of reducing power infermentation or enzymatic reactions and that can use electron mediatorsand a biocatalyst, such as cells or enzymes, to produce electricity.

[0005] 2. Description of the Related Art

[0006] A biofuel cell is a device that directly converts microbialmetabolic power into electricity using electrochemical technology. (See,for example, Allen, “Cellular Electrophysiology”, p. 247-283, In J. R.Norris and D. W. Ribbons (eds.). Methods in Microbiology. AcademicPress, New York, 1992; Bennetto, et al. “The Sucrose Fuel Cell:Efficient Biomass Conversion Using A Microbial Catalyst”, Biotechnol.Lett. 7:699-105, 1985; Roller et al., “Electron-Transfer Coupling InMicrobial Fuel Cells: 1. Comparison Of Redox-Mediator Reduction RatesAnd Respiratory Rates Of Bacteria”, J. Chem. Tech. Biotechnol. 34B:3-12,1984; and Thurston, et al., “Glucose Metabolism In A Microbial FuelCell. Stoichiometry Of Product Formation In A Thionine-Mediated ProteusVulgaris Fuel Cell And Its Relation To Coulombic Yields”. J. Gen.Microbial. 131:1393-1401,1985.). Chemical energy is converted toelectric energy by coupling the biodegradative oxidation of organic orinorganic substrates to the chemical reduction of an oxidant at theinterface between the anode and the cathode (see, Willner et al. “ABiofuel Cell Based On Pyrroloquinoline Quinone And Microperoxidase-11Monolayer-Functionalized Electrodes”, Bioelectrochem. Bioenerg,44:209-214, 1998.). Direct electron transfer from microbial cells toelectrodes occurs at very low efficiencies (See, Allen, “CellularElectrophysiology”, p. 247-283, In J. R. Norris and D. W. Ribbons(eds.). Methods in Microbiology. Academic Press, New York, 1992). Inmicrobial fuel cells, two redox couples are required, one for couplingreduction of an electron mediator to bacterial oxidative metabolism, andthe other for coupling oxidation of the electron mediator to thereduction of the electron acceptor on the cathode surface where theelectron acceptor is regenerated with atmospheric oxygen (see,Ardeleanu, et al., “Electrochemical Conversion In Biofuel Cells UsingClostridium Butyricum Or Staphylococcus Aureus Oxford”, Bioelectrochem.Bioenerg, 11:273-277, 1983; and Delaney, et al., “Electron-TransferCoupling In Microbial Fuel Cells. 2. Performance Of Fuel CellsContaining Selected Microorganism-Mediator-Substrate Combinations”,Chem. Tech. Biotechnol. 34b:13-27,1985).

[0007] Electron transfer from a microbial electron carrier to anelectrode requires an electron mediator (See, Fultz et al., “MediatorCompounds For The Electrochemical Study Of Biological Redox Systems: ACompilation”, Anal. Chim. Acta. 140:1-18, 1982.). Previous studiesreported that metabolic reducing power produced by Escherichia coli orProteus vulgaris was converted to electricity by using mediators such as2-hydroxy-1,4-naphtoquinone (HNQ) or thionin (see, Tanaka et al.,“Effects Of Light On The Electrical Output Of BioelectrochemicalFuel-Cells Containing Anabaena Varibilis M-2: Mechanisms Of The PostIllumination Burst”, Chem. Tech. Biotechnol. 42:235-240,1988; and Tanakaet al., “Bioelectrochemical Fuel-Cells Operated By The Cyanobacterium,Anabaena Variabilis”, Chem. Tech. Biotechnol. 35B: 191-197, 1985). Parket al. in “Electrode Reaction Of Desulfovibrio Desulfuricans ModifiedWith Organic Conductive Compounds”, Biotech. Techniq. 11:145-148, 1997confirmed that viologen dyes (see, Kim et al., “Benzyl Viologen CationRadical: First Example Of A Perfectly Selective Anion lonophore Of TheCarrier Type”, Biochem. Biophys. Res. Com., 180:11276-1130,1982; andMorimyo, “Isolation And Characterization Of Methyl Viologen SensitiveMutants Of Escherichia Cofi K-12”, J. Bactedol. 170:2136-2142, 1988)cross-linked with carbon polymers and absorbed to Desulfovivriodesulfuricans cell membranes can mediate electron transfer toelectrodes. Kim et al. in “Direct Electrode Reaction Of Fe(III)-ReducingBacterium, Shewanella Putrefacians” J. Microbial. Biotechnol., 9:127-13,1999 showed that Shawella putrefacians, which contains outer-membranecytochromes able to reduce Fe³⁺, was electroactive and, that it couldgrow on lactate as the E electron donor with a graphite felt electrodeas the electron acceptor in a complex biofuel cell. U.S. Pat. No.6,270,649 to Zeikus et al. shows that neutral red is an improvedelectron mediator for either converting electricity into microbialreducing power for enhanced cell growth and production of reducedend-products (see, Park et al., “Microbial Utilization Of ElectricallyReduced Neutral Red And The Sole Electron Donor For Growth AndMetabolite Production”, Appl. Environ. Microbiol. 65:2912-2917, 1999;and Park et al., “Utilization Of Electrically Reduced Neutral Red ByActinobacillus Succinogenes: Physiological Function Of Neutral Red InMembrane-Driven Fumarate Reduction And Energy Conservation”, J.Bacteriol. 1812:2403-2410, 1999), or converting microbial reducing powerinto electricity in biofuel cells (see, Park and Zeikus, “ElectricityGeneration In Microbial Fuel Cells Using Neutral Red And AnElectronophore”, Appl. Environ. Microbiol. 66:1292-1297, 2000). Park etal. in “Electricity Production In Biofuel Cell Using Modified GraphiteElectrode With Neutral Red”, Biotech. Lett. 22:1301-1304, 2000 showedthat binding neutral red to a graphite electrode further enhancedelectron transfer efficiency in microbial fuel cells.

[0008] The electrical enhancement of fermentations andbiotransformations also involves the utilization of an electrode andelectron mediator in a bioreactor system which overproduces reduced endproducts (see, Hongo et al., “Application Of Electro-Energizing MethodTo L-Glutamic Acid Fermentation”, Agri. Biolio. Chem., 43: 2075-208111979; Hongo et al., “Application Of Electro-Energizing Method ToL-Glutamic Acid Fermentation”, Agri. Biolio. Chem., 43: 2083-2086, 1979;Kim et al., “Electron Flow Shift In Clostridium AcetobutylicumFermentation By Electrochemically Introduced Reducing Equivalent” 1988;Park and Zeikus “Utilization Of Electrically Reduced Neutral Red ByActinobacillus Succinogenes: Physiological Function Of Neutral Red InMembrane-Driven Fumarate Reduction And Energy Conservation”, J.Bacteriol. 181: 403-2410, 1999; and Shin et al., “Evaluation Of AnElectrochemical Bioreactor System In The Biotransformation Of6-Bromo-2-Tetralone To 6-Bromo-2-Tetralol”, Appl Microbiol Biotechnol.,DOI 10.1007/s002530100809. Online publication: Sep. 22, 2001.) Forexample, a graphite felt electrode and soluble neutral red can greatlyenhance the yields of succinate produced by fermentation (see Park andZeikus “Utilization Of Electrically Reduced Neutral Red ByActinobacillus Succinogenes: Physiological Function Of Neutral Red InMembrane-Driven Fumarate Reduction And Energy Conservation”, J.Bactedol. 181: 403-2410, 1999) and, tetralol produced by yeasttransformation (Shin et al., “Evaluation Of An ElectrochemicalBioreactor System In The Biotransformation Of 6-Bromo-2-Tetralone To6-Bromo-2-Tetralol”, Appl Microbiol Biotechnol., DOI10.1007/s002530100809. Online publication: Sep. 22, 2001). Neutral redworks in part by direct chemical reduction of pyridine nucleotides (Parkand Zeikus “Utilization Of Electrically Reduced Neutral Red ByActinobacillus Succinogenes: Physiological Function Of Neutral Red InMembrane-Driven Fumarate Reduction And Energy Conservation”, J.Bacteriol. 181: 403-2410,1999).

[0009] The use of oxidoreductases in microbial electrochemical cells hasalso been proposed. One major factor limiting the utilization ofoxidoreductases in chemical syntheses (see, e.g., S. M. Roberts et al.,Chimicaoggi, “Some Recent Advances In The Synthesis Of Optically PureFine Chemicals Using Enzyme-Catalyzed Reactions In The Key Step”,July/August 1993, pp. 93-104; and D. Miyawaki et al., “ElectrochemicalBioreactor With Immobilized Glucose 6-Phosphate Dehydrogenase On TheRotation Graphite Disc Electrode Modified With Phenazine Methosulfate”,Enzg. Microbiol. Technol. 15:525-29,1993) or in chemical detection,i.e., biosensors (see, e.g., P. N. Bartlett, “Modified Electrode SurfaceIn Amperometric Biosensors”, Med. and Biol. Eng. and Comput. 28: B10-B7,1990; and D. Miyawaki et al., supra) is the lack of a-facile system forregeneration or recycling of the electron transferring cofactors (e.g.,nicotinamide adenine dinucleotide, quinones, flavin adeninedinucleotide, etc).

[0010] It has been reported by Park and Zeikus in “Utilization OfElectrically Reduced Neutral Red By Actinobacillus Succinogenes:Physiological Function Of Neutral Red In Membrane-Driven FumarateReduction And Energy Conversion”, J. Bacteriol. 181:2403-2410, 1999 thatneutral red would undergo reversible chemical oxidoreduction withnicotinamide adenine dinucleotide (i.e., recycle nicotinamide adeninedinucleotide electrochemically). It has also been reported that by usingsoluble neutral red in electrochemical reactors containing microbesthat: (1) microbes could grow on electricity alone; (2) diverse microbescould over-produce a variety of reduced biochemicals duringfermentations of biotransformations; and (3) microbes could generateelectricity during digestion of organic matter. (See, e.g., Park et al.,“Microbial Utilization Of Electrically Reduced Neutral Red In The SoleElectron Donor For Growth And Metabolite Production”, Appl. Environ.Microbiol. pp. 2912-2917, 1990; Park and Zeikus, “Electricity GenerationIn Microbial Fuel Cells Using Neutral Red As An Electronophore”, Appl.Environ. Microbiol., 66:1292-1297, 2000; and U.S. Pat. No. 6,270,649).

[0011] Because of the importance of electrodes and electron mediators inbioreactor systems for electricity generation, chemical sensing, andelectrical enhancement of chemical synthesis, fermentations andbiotransformations, there is a continuing general need for improvedelectrodes that enhance the rate of electron transfer from cells.Preferably, the improved electrode compositions for increased electrontransfer efficiency can use resting cells from pure and mixed bacterialcultures. In one specific application, there is a need for an improvedelectrode that has utility as an enzymatic fuel cell, as a sensor forsuccinate detection, and as an engineered catalyst for the synthesis offumarate or succinate. In particular, there is a need for an enzymeimmobilization protocol to link nicotinamide adenine dinucleotide (NAD),neutral red (NR), and fumarate reductase to an electrode in anelectrochemical reactor.

[0012] Microbial electrochemical cells have previously usedtwo-compartment systems whereby the aerated cathode compartment containsa chemical solution of ferric cyanide and oxygen, and the anodecompartment contains bacterial cells, electron mediator, and reducedsubstrate (see, for example, Ardeleanu, et al., “ElectrochemicalConversion In Biofuel Cells Using Clostridium Butyricum OrStaphylococcus Aureus Oxford”, Bioelectrochem. Bioenerg, 11:273-277,1983; and Park and Zeikus, “Electricity Generation In Microbial FuelCells Using Neutral Red And An Electronophore”, Appl. Environ.Microbiol. 66:1292-1297, 2000). Two compartment fuel cells are generallynot practical because of the requirement for a ferricyanide solution andaeration in the cathode compartment. Thus, there is also a need for asingle compartment microbial electrochemical cell that eliminates therequirements for a ferricyanide solution and aeration in the cathodecompartment.

SUMMARY OF THE INVENTION

[0013] The foregoing needs are met by an electrochemical bioreactorsystem according to the invention. The electrochemical bioreactor systemcomprises a first compartment containing a first electrode; a secondelectrode; an electrically conductive material connecting the firstelectrode and the second electrode; an electrolyte for providing ionicconductivity between the first electrode and the second electrode; and abiocatalyst disposed in the first compartment or associated with thefirst electrode.

[0014] When an electrical power supply is electrically connected to theelectrically conductive material and electrical current is applied fromthe electrical power supply to the first electrode and the secondelectrode,: the first electrode acts as a cathode whereby theelectrochemical bioreactor system provides electrical enhancement ofchemical synthesis and/or fermentations and/or biotransformationsoccurring in the first compartment. When an electrical load (e.g., aresistive element) is electrically connected to the electricallyconductive material, the first electrode acts as a anode and theelectrochemical bioreactor system generates electrical currentdetectable at the load from materials in the first compartment. Theelectrical current detectable at the load may be used as a source ofelectricity, or measurement of the electrical current at the load mayused in chemical or biochemical sensing devices.

[0015] In a first aspect of the invention, the first electrode of theelectrochemical bioreactor system comprises graphite felt and at leastone electron mediator associated with the graphite felt. As used herein,a first material (in the first aspect of the invention, an electronmediator) is “associated” with a second material (in the first aspect ofthe invention, graphite felt) if the first material is directly orindirectly, physically or chemically bound to the second material. Afirst material may be physically bound to a second material byentrapping, imbedding or otherwise containing the first material withinthe second material. A first material may be chemically bound to thesecond material by way of a chemical reaction wherein the first materialis covalently or ionically bonded to the second material. Thus, varioustechniques for associating at least one electron mediator with thegraphite felt are contemplated herein.

[0016] In one version of the first aspect of the invention, the firstelectrode comprises neutral red (NR) bound to the graphite felt. Inanother version of the first aspect of the invention, the firstelectrode comprises a carboxylated cellulose (e.g.,carboxymethylcellulose) bound to the graphite felt and neutral red boundto the carboxylated cellulose. In yet another version of the firstaspect of the invention, the first electrode comprises NAD⁺ bound to thegraphite felt. In still another version of the first aspect of theinvention, the first electrode comprises a carboxylated cellulose boundto the-graphite felt and NAD⁺ bound to the carboxylated cellulose. In afurther version of the first aspect of the invention, the firstelectrode comprises a carboxylated cellulose bound to the graphite felt,neutral red bound to the carboxylated cellulose, NAD⁺ bound to thegraphite felt, and an oxidoreductase (e.g., fumarate reductase) bound tothe graphite felt. Each of these versions of the first electrode servesto enhance the rate of electron transfer from the biocatalyst.

[0017] In a second aspect of the invention, the first electrode of theelectrochemical bioreactor system comprises a metal ion electronmediator. For example, the first electrode may comprise an iron cation(e.g., Fe³⁺) and/or a manganese cation (e.g., Mn⁴⁺) associated with agraphite plate. Each of these versions of the second electrode serves toenhance the rate of electron transfer from the biocatalyst.

[0018] Various biocatalysts are suitable for use in an electrochemicalbioreactor system in accordance with the invention. For example, thebiocatalyst may comprise an oxidoreductase (e.g., fumarate reductase)bound to the first electrode. The biocatalyst may also comprisebacterial cells disposed in the first compartment of the electrochemicalbioreactor system. Non-limiting examples of bacterial cells includecells of Actinobacillus succinogenes, cells of Escherichia coli, andsewage sludge.

[0019] In a third aspect of the invention, the electrochemicalbioreactor system is configured as a single compartment electrochemicalcell. In the third aspect of the invention, the second electrodeincludes a first surface-and a second opposed surface and theelectrolyte is disposed on the first surface of the second electrode.The first surface faces an interior of the first compartment of theelectrochemical bioreactor system and the second surface of the secondelectrode comprises an exterior surface of the first compartment of theelectrochemical bioreactor system. The single compartmentelectrochemical cell design eliminates the requirements for aferricyanide solution and aeration in the cathode compartment. When thesingle compartment electrochemical cell is used as an electricitygenerator, bacteria attach to the anode and electrons are transferredfrom the cells metabolic pool to reduce either neutral red or a metallicelectron mediator immobilized on the anode. The electron driving forcegenerated is coupled to reduction of Fe ions on the cathode which aresubsequently oxidized by O₂ in the air. Consequently, a catholytesolution and aeraton are not required.

[0020] It is therefore an advantage of the present invention to providean electrochemical bioreactor system having improved electrodes thatenhance the rate of electron transfer from cells.

[0021] It is another advantage of the present invention to provide anelectrochemical bioreactor system having an improved electrode that hasutility as an enzymatic fuel cell.

[0022] It is yet another advantage of the present invention to providean electrochemical bioreactor system having an improved electrode thathas utility as a sensor for succinate detection.

[0023] It is still another advantage of the present invention to providean electrochemical bioreactor system having an improved electrode thathas utility as an engineered catalyst for the synthesis of fumarate orsuccinate.

[0024] It is a further advantage of the present invention to provide anelectrochemical bioreactor system having an improved electrode with anenzyme immobilization protocol to link nicotinamide adenine dinucleotide(NAD⁺), neutral red, and fumarate reductase to the electrode.

[0025] It is yet another advantage of the invention to provide a fuelcell system that can be used as either an enrichment device forelectrophilic microorganisms; that is, those which use an electrode asan electron acceptor, or electron donor for energy conservation.

[0026] These and other features, aspects, and advantages of the presentinvention will become better understood upon consideration of thefollowing detailed description, drawings, and appended claims.

DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 is a schematic structure of a neutral red woven graphiteelectrode according to a first aspect of the invention. Neutral red waslinked by a covalent bond between its amine and the carboxy of wovengraphite.

[0028]FIG. 2 shows the electricity production (in solid symbols) coupledto oxidation of NADH to NAD⁺ on a normal electrode and electricityproduction coupled to oxidation of NADH to NAD⁺ (in open symbols) on amodified electrode with Mn according to a second aspect of theinvention. The theoretical efficiency of electricity production on thenormal electrode and the modified electrode was 26.442% and 36.67%,respectively.

[0029]FIG. 3 is a schematic diagram of a single compartment fuel cellaccording to a third aspect of the invention depicting electron transferfrom cell metabolism to the anode metals to the cathode metals; and,proton transfer through the porcelain septum to water. The electrondriving force is produced by the potential difference between the mostoxidized metallic ion in the anode to the most reduced metallic ion inthe cathode.

[0030]FIG. 4 is a diagrammatic representation of the single compartmentfuel cell of FIG. 3. The single compartment fuel cell comprises a Pyrex™glass container (total volume 500 ml.) with a Fe³⁺ cathode (50 cm²surface area) and a rubber bung with a connected Mn⁴⁺ graphite anode andan N₂ gassing port.

[0031]FIG. 5 is a comparison of electrical current and potential levelsobtained when Escherichia coli was used in a two compartment fuel cell(A, B and C) versus a single compartment fuel cell (D, E and F). Threedifferent types of anode and cathode combinations were applied to eachfuel cell system. (A) and (D) were a woven graphite anode and a, Fe³⁺graphite cathode; (B) and (E) were a neutral red woven graphite anodeand a Fe³⁺ graphite cathode; and (C) and (F) were a Mn⁴⁺ graphite anodeand a Fe³⁺ graphite cathode.

[0032]FIG. 6 is a comparison of electrical current and potential levelsobtained when sewage sludge was used in a two compartment fuel cell (A,B and C) versus a single compartment fuel cell (D, E and F). Threedifferent types of anode and cathode combinations were applied to eachfuel cell system. (A) and (D) were a woven graphite anode and a Fe³⁺graphite cathode; (B) and (E) were a woven graphite anode with neutralred and a Fe³⁺ graphite cathode; and (C) and (F) were a Mn⁴⁺ graphiteanode and a Fe³⁺ graphite cathode.

[0033]FIG. 7 is a scanning electron microscope photograph of grazingmicrobes attached to a neutral red -woven graphite anode. Theseelectrogenic microbes were predominantly cocci that were enriched fromsewage sludge.

[0034]FIG. 8 is a schematic diagram for depicting electricity generationon different electrode combinations during electron transfer frommicrobes to oxygen and protons via the anode (left) to the cathode(right). In System 1, the electrons are difficult to transfer from themicrobial cells to the anode, the cathode, and oxygen because there isneither an electron channel (i.e. an electron mediator), nor a highoxygen affinity metallic ion in the cathode. Consequently, the currentobtained in System 1 is low but the potential difference between theanode and cathode is high. In System 2, the electrons are difficult totransfer from the microbes to the anode but are easier to transfer fromthe cathode to oxygen because of Fe³⁺; thus, the current produced ishigher than System 1 but the potential is similar. In System 3, theelectrons are easy to transfer from the microbes to the anode andcathode because of neutral red and Fe³⁺; thus, the current is higher butthe potential is lower in System 1 or 2. In System 4, the electrons arevery easy to transfer from the microbes to the anode and cathode becauseof Mn⁴⁺ as an electron mediator and Fe³⁺ as a high oxygen affinity ion;thus, the current obtained is higher than in System 1 or 2, but thepotential is lower than in System 1, 2 or 3 because the potentialdifference between Mn⁴⁺ and Fe³⁺ is lower than between neutral red andFe³⁺ or no electron mediator and Fe The E_(o)′ value (in volts) forelectron mediators are: neutral red −0.325; Mn +0.35: Fe, +0.78; and O₂,+0.82. The cytoplasmic membrane functions are an electron barrierproviding resistance to electron transfer which can be overcome byadding neutral red or Mn⁴⁺ which serves as an electron mediator orchannel.

[0035]FIG. 9 is a schematic diagram showing one compartment of abioelectrochemical cell using a graphite felt electrode modified withCMC-NR-NAD⁺-fumarate reductase complex according to the invention.

[0036]FIG. 10 is a schematic diagram showing a graphite felt electrodemodified with CMC-NR-NAD⁺-fumarate reductase complex according to theinvention.

[0037]FIG. 11 is a schematic diagram showing the mechanisms foroxidation of succinate to fumarate coupled to electricity production andthe reduction of fumarate to succinate coupled to electricityconsumption using an electrode modified with CMC-NR-NAD⁺-fumaratereductase complex according to the invention.

[0038]FIG. 12 is a graph showing succinate detection by abioelectrochemical cell using a graphite felt electrode modified withCMC-NR-NAD⁺-fumarate reductase complex according to the invention.

[0039]FIG. 13 is a graph showing the potentiometric and amperometricresponse of a bioelectrochemical cell using a graphite felt electrodemodified with CMC-NR-NAD⁺-fumarate reductase complex according to theinvention.

[0040]FIG. 14 is a graph showing the oxidation of succinate to fumarateon a graphite felt electrode modified with CMC-NR-NAD⁺-fumaratereductase complex according to the invention.

[0041]FIG. 15 is a graph showing the reduction of fumarate to succinateon a graphite felt electrode modified with CMC-NR-NAD⁺-fumaratereductase complex according to the invention.

[0042]FIG. 16 shows that a Mn⁺⁴ graphite electrode according to theinvention can electrochemically reduce NAD⁺ to NADH. FIG. 16 depicts theelectrochemical reduction of NAD⁺ to NADH by a Mn(IV)-graphite cathodeand a Fe(III)-graphite anode. A 1 mM NAD⁺ solution in Tris—HCl buffer(100 mM, pH 7.0) was the catholyte and a 200 mM KH₂PO₄ solution indeionized distilled water was the anolyte. Line 1 is the spectrum of 0.5mN NADH; Line 2 is the spectrum of NADH reduced by the Mn(IV)-graphiteelectrode; and Line 3 is the spectrum of 1 mM NAD⁺ that is not reducedby a normal graphite felt electrode.

[0043]FIG. 17 shows a controlled fumarate fermentation by A.succinogenes without electricity, that is a reduction of fumarate tosuccinate by growing cells of Actinobacillus succinogenes in abioreactor without electrochemical reaction.

[0044]FIG. 18 shows the reduction of fumarate to succinate by growingcells of Actinobacillus succinogenes in an electrochemical reactor usinga Mn(IV)-graphite electrode as the cathode. A Fe(III)-graphite electrodewas used as the anode. The density of the binding cell to anode was 0.12as OD₆₆₀. The binding bacterial cells to electrode were calculated usinga predetermined calibration curve (bacterial density (OD₆₆₀)=bacterialprotein concentration (mg/ml)×1.7556). Protein of the binding bacterialcell to electrode was obtained by the alkaline lysis method (see, Parket al., “Utilization Of Electrically Reduced Neutral Red ByActinobacillus Succinogenes: Hysiological Function Of Neutral Red InMembrane-Driven Fumarate Reduction And Energy Conservation”, J.Bacterol. 1812:2403-2410, 1999).

[0045]FIG. 19 shows the effect of substrate concentration on thebioelectrochemical reduction of fumarate to succinate. Resting cells ofActinobacillus succinogenes were used. The cell density was adjusted to4.0 (OD₆₆₀). The Fe(III)-graphite and Mn(IV)-graphite were used as anode(160 cm² surface area) and a cathode (160 cm² surface area)respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention is directed to electrodes andconfigurations for electrochemical bioreactor systems that can useelectrical energy as a source of reducing power in fermentation orenzymatic reactions and that can use electron mediators and abiocatalyst, such as cells or enzymes, to produce electricity. Theelectrochemical bioreactor system comprises a first compartmentcontaining a first electrode; a second electrode; an electricallyconductive material connecting the first electrode and the secondelectrode; an electrolyte for providing ionic conductivity between thefirst electrode and the second electrode; and a biocatalyst disposed inthe first compartment or associated with the first electrode.

[0047] When an electrical power supply is electrically connected to theelectrically conductive material and electrical current is applied fromthe electrical power supply to the first electrode and the secondelectrode, the first electrode acts as a cathode whereby theelectrochemical bioreactor system provides electrical enhancement ofchemical synthesis and/or fermentations and/or biotransformationsoccurring in the first compartment. When an electrical load (e.g., aresistive element) is electrically connected to the electricallyconductive material, the first electrode acts as a anode and theelectrochemical bioreactor system generates electrical currentdetectable at the load from materials in the first compartment. Theelectrical current detectable at the load may be used as a source ofelectricity, or measurement of the electrical current at the load mayused in chemical or biochemical sensing devices.

[0048] In one form, the first electrode comprises neutral red covalentlybound to graphite felt. It has been discovered that the efficiency ofelectron transfer from microbial cells to electrodes can be increased bycovalently linking neutral red to woven graphite felt. Neutral red canbe covalently linked to graphite felt by converting at least a portionof the surface of the graphite felt to its carboxy form (using heat, forexample) and covalently linking the amine group of neutral red to thecarboxy of the woven graphite. FIG. 1 shows the covalent linking ofneutral red to the graphite felt. The neutral red is immobilized on thegraphite felt and does not leach-out in water.

[0049] The first electrode comprising neutral red covalently bound tographite felt may then be incorporated into an electrochemicalbioreactor system using known methods within the ability of one skilledin the art. A typical electrochemical bioreactor system will include twocompartments, each of which contains one of the first and the secondelectrodes. The second electrode may comprise an iron cation (e.g.,Fe³⁺) associated with a graphite plate. The electrodes are separated byan lonically conductive electrolyte material between the first electrodeand the second electrode. Typically, the electrolyte allows for thepassage of protons and cations only. Solid electrolyte materials arewell known and include materials such as Nafion™ cationic selectivemembranes and porcelain septums. Catholytes and anolytes may also beused in the compartments. For example, catholytes and anolytes that havebeen found to be suitable in electrochemical bioreactors includebacterial growth media or a phosphate buffer. The electrodes can beconnected to an electrical power source or to a multimeter (or otherresistive load) using an electrically conductive material such as ametallic material (e.g., platinum wire). A suitable biocatalyst isdisposed in the compartment containing the first electrode or isassociated (as defined above) with the first electrode.

[0050] A wide range of biocatalysts to promote cell growth or theformation of reduced products in the electrochemical bioreactor systemcan be used. For example, a wide variety of bacteria, archea, plantcells or animal cells can be used. Non-limiting examples include cellsof Actinobacillus succinogenes, cells of Escheichia coli, and sewagesludge. Enzyme preparations may also be used in the practice of theinvention. A desired enzyme may be partially purified using standardmethods known to one of ordinary skill in the art. The enzyme may beisolated from its native source or from a transgenic expression host, orobtained through a commercial vendor. Useful enzymes include any enzymethat can use reducing power from an electron mediator to form a desiredreduced product, or which can transfer reducing power to an electronmediator and form a desired oxidized product. Most commonly, thisreduction is mediated by NADPH or NADH. An oxidoreductase may be used asthe biocatalyst in the practice of the invention. For example, isolatedalcohol dehydrogenases, carboxylic acid reductase, and fumaratereductase could be used in the electrochemical bioreactor system.

[0051] In another form, the first electrode of the electrochemicalbioreactor system comprises a metal ion electron mediator. For example,the first electrode may comprise an iron cation (e.g., Fe³⁺) and/or amanganese cation (e.g., Mn⁴⁺) associated with a graphite plate. Thefirst electrode comprising the metal ion electron mediator may beincorporated into. an electrochemical bioreactor system using knownmethods as described above.

[0052] The present invention also provides a method for utilizingoxidoreductase as biocatalysts for chemical sensing and chemicalproduction, and as a biofuel cell by immobilizing on an electrode anoxidoreductase and multiple electron carriers such as nicotinamideadenine dinucleotide (NAD⁺) and neutral red (NR) which can bebioelectrically regenerated. In one version of the invention, fumaratereductase enzyme is immobilized onto a graphite felt electrode that ismodified with carboxymethylcellulose (CMC), neutral red (NR) andnicotinamide adenine dinucleotide (NAD⁺). The fumarate reductase enzymeis immobilized onto a CMC-NR-NAD⁺ modified graphite felt electrode bypreparing fumarate reductase, preparing a graphite electrode substrate,treating the electrode with neutral red, treating the electrode withcarboxymethylcellulose, treating the electrode with NAD⁺, and treatingthe electrode with the fumarate reductase to produce aCMC-NR-NAD⁺-fumarate reductase modified electrode.

[0053] Looking at FIG. 10, there is shown schematically a graphite feltelectrode with a CMC-NR-NAD⁺-fumarate reductase complex according to theinvention. The chemical linkings between carboxymethylcellulose, NAD⁺,fumarate reductase and neutral red on the graphite electrode aredepicted in FIG. 10. The CMC-NR-NAD⁺-fumarate reductase complex is athin film on the electrode. The graphite felt electrode is modified withneutral red before coating with the CMC-NR-NAD⁺-fumarate reductasecomplex. The electrons can be reversibly transferred from the graphitefelt to the fumarate reductase or from the fumarate reductase to thegraphite felt by the coupling oxidation-reduction reaction of neutralred and NAD⁺. The coupling oxidation-reduction reaction of neutral redand NAD⁺ provides a mechanism by which fumarate can be reduced tosuccinate thereby consuming electricity and succinate can be oxidized tofumarate thereby producing electricity.

[0054] The CMC-NR-NAD⁺-fumarate reductase electrode may be incorporatedinto an electrochemical bioreactor system for chemical sensing, chemicalproduction and electricity production as shown schematically in FIG. 9.FIG. 9 illustrates an electrochemical bioreactor system that may be usedfor succinate detection or fumarate synthesis. The anode containsfumarate reductase, NAD⁺, and neutral red immobilized on a graphiteelectrode with carboxymethylcellulose according to the invention.

[0055] Turning to FIG. 11, there is shown a schematic that depicts howthe CMC-NR-NAD⁺-fumarate reductase enzyme immobilized onto the graphitefelt electrode can function as a fuel cell during succinate oxidationand as a catalyst producing succinate from electricity and fumarate. Theoxidation of succinate to fumarate coupled to electricity production andthe reduction of fumarate to succinate coupled to electricityconsumption is catalyzed by fumarate reductase which is bound tocarboxymethylcellulose with NAD⁺ and neutral red. The graphite feltelectrode is coated with the CMC-NR-NAD⁺-fumarate reductase complex. Theneutral red, NAD⁺ and the fumarate reductase can be covalently bonded tothe carboxy residues of both the graphite felt and thecarboxymethylcellulose.

[0056] The fumarate reductase is used as a oxidoreductase for thecontinuous enzymatic reduction-oxidation during bioelectrochemicalsynthesis or the detection of chemicals. When the fumarate reductaseenzyme is immobilized onto a graphite felt electrode that is modifiedwith carboxymethylcellulose (CMC), neutral red (an electron mediator)and nicotinamide adenine dinucleotide, the detection of succinate withthis bioelectrode is linear from a lower level of 5 mM to 10 mM. Thesynthesis of fumarate using this bioelectrode is dependent on succinateutilization and results in a proportional amount of electricity andfumarates produced. The synthesis of succinate using the bioelectrode isalso dependent on current and fumarate concentration. Thiselectrochemical bioreactor system can enhance the utility ofoxidoreductases in diverse enzymatic fuel cells, chemical synthesis andchemical detection.

[0057] It has been discovered that the above electrodes can beincorporated into a single compartment electrochemical bioreactor systemaccording to the invention. The single compartment electrochemicalbioreactor system is shown in FIGS. 3 and 4. FIG. 3 provides a schematicof how the single compartment fuel cell works. Bacteria attach to theanode and electrons are transferred from the cells metabolic pool toreduce either neutral red or a metallic electron mediator immobilized onthe anode. The electron driving force generated is coupled to reductionof Fe ions on the cathode which are subsequently oxidized by O₂ in air.Thus, a catholyte solution and aeration is not required. Protons aretransferred from the anolyte solution through the micropore system onthe cathode.

EXAMPLES

[0058] The following examples serve to further illustrate the invention.The examples are not intended to limit the invention in any way.

Example 1 Microbial Preparations And Measurements

[0059] Sewage sludge (i.e., a mixture of anaerobic bacteria) wasobtained from the Jackson, Michigan, USA, sewage treatment plant. Thefresh anaerobic sludge was allowed to settle under an N₂ atmosphere for1 day to remove solid particles. The suspended cells were used as acatalyst and were maintained in an anaerobic bottle by adding glucose (5g/L) once a week. The resting cells were harvested by centrifugation at5,000×9 at 4° C. and were washed twice and resuspended in anolyte mediumI. Anolyte medium I contained: 100 mM phosphate buffer (pH 7.0), 10 g/Lsodium lactate, 5 g/L peptone and 5 g/L yeast extract. The initialconcentration of sewage sludge cell suspension was adjusted to 5.5 mg/mlcell protein as determined (see, Park and Zeikus, “Utilization OfElectrically Reduced Neutral Red By Actinobacillus Succinogenes:Physiological Function Of Neutral Red In Membrane-Driven FumarateReduction And Energy Conservation”, J. Bacteriol. 1812:2403-2410, 1999)using Bradford reagent and bovine serum albumen as standard.

[0060]Escherichia coli K12 was grown anaerobically under N₂ gas for 20hours to the stationary phase in an LB medium which contained: 10 g/Lpeptone, 5 g/L yeast extract and 10 g/L NaCl. The resting cells wereharvested at 4° C. by centrifugation at 5,000×g. The resting cells werewashed twice and resuspended in Anolyte medium I at an optical densityof 3.5 at 660 nm which corresponded to a 4.73 mg/ml cell protein.

Example 2 Electrode Compositions

[0061] A procedure to covalently link neutral red to woven graphite feltwas developed (see FIG. 1). The theoretical surface area of the wovengraphite felt anode is 1.27 m²(2.7 g) versus a graphite plate anode withapproximately 80 cm². This procedure involved the following steps: (1.)cleaning the graphite felt electrode by soaking in methanol for 12 hoursand then distilled water for 12 hours; (2.) drying the electrode at 120°C. for 1 hour;-(3) making a carboxy surface by heating at 200° C. for 48hours; (4) soaking the electrode in dicyclohexylcarbodimide solution (2mg/ml chloroform) at 4° C. for 6 hours; (5) binding neutral red (100μmol) to the electrode by incubating the dicyclohexylcarbodimidesolution at 4° C. for 12 hours. The neutral red was immobilized to theelectrode by this procedure and did not leach-out in water.

[0062] Several different metallic graphite electrodes were designed soas to compare their electron transfer efficiencies to neutralred-graphite felt. A ferric (Fe⁺³) graphite electrode was made by mixingferric sulfate (3% w/w) with 60% (w/w) fine graphite powder (below 500mesh), 36% (w/w) kaolin (porcelain) with a particle size below 400 meshand 1.0% (w/w) nickel chloride. One part distilled water and 2 parts ofthis mixture were stirred into a paste and molded into a 20 cm×20 cm×1cm thick plate by pressing at 1.0 kg/cm², drying in air for 48 hours atroom temperature, and then baking at 1100° C. for 12 hours in a kilnkept anaerobic with a flow of N₂ gas. A Mn⁺⁴ graphite electrode was madein the same manner except 3% w/w manganese sulfate replaced the ferroussulfate.

[0063] The cathodes differed from the anodes in the single compartmentfuel cell because the inside of the cathode was coated with a 1millimeter thickness porcelain septum made from 100% kaolin. Theporcelain septum enabled protons to transfer from the anolyte to thecathode.

Example 3 Fuel Cell Design And Operation

[0064] Two compartment cell fuel cells were prepared using theconfiguration described in Park and Zeikus, “Electricity Generation InMicrobial Fuel Cells Using Neutral Red And An Electronophore”, Appl.Environ. Microbiol., 66:1292-1297, 2000, except for using the electrodesprepared in Example 2; and the cation selective membrane was replacedwith a 50 mm by 2 mm thick porcelain septum made from 100% Kaolin asdescribed above in Example 2. The two compartment fuel cell of Park andZeikus, “Electricity Generation In Microbial Fuel Cells Using NeutralRed And An Electronophore”, Appl. Environ. Microbiol., 66:1292-1297,2000 requires aeration and ferricyanide solution in the cathodecompartment.

[0065] Because two compartment fuel cells are generally not practicalbecause of the requirement for a ferricyanide solution and aeration inthe cathode compartment, single compartment fuel cell design as shown inFIGS. 3 and 4 was prepared in order to eliminate the requirements for aferricyanide solution and aeration in the cathode compartment. FIG. 3provides a schematic of how the single compartment fuel cell works.Bacteria attach to the anode and electrons are transferred from thecells metabolic pool to reduce either neutral red or a metallic electronmediator immobilized on the anode. The electron driving force generatedis coupled to reduction of Fe ions on the cathode which are subsequentlyoxidized by O₂ in the air. Consequently, a catholyte solution andaeration is not required. Protons are transferred from the anolytesolution through the micropore system on the cathode. A Mn⁴⁺ graphiteanode and an Fe³⁺ graphite cathode achieved the highest level of current(i.e. ˜14 mA) using sewage sludge microbes. This was four-fold higherthan the best E-coli value obtained and, it was three-fold higher thanwhat was previously described for sewage sludge using soluble neutralred and a plain, woven graphite electrode (See, Park and Zeikus,“Electricity Generation In Microbial Fuel Cells Using Neutral Red And AnElectronophore”, Appl. Environ. Microbiol., 66:1292-1297, 2000).

[0066] Resting cell suspensions in anolyte medium I were placed in theanoxic anode compartment of the two versus one compartment fuel cellsystems and electrical current and potential were measured as reportedin Park and Zeikus, “Electricity Generation In Microbial Fuel CellsUsing Neutral Red And An Electronophore”, Appl. Environ. Microbiol.,66:1292-1297, 2000. Experiments compared electrical performance of E.coli versus sewage sludge in two versus one compartment fuel cells thatcontained different electrode compartments for the anode and cathode.The electrical measurements used a joule as the unit of energy which wascalculated using the equation; ampere (A) times volt (V) times time(sec). A coulomb is equal to A times sec, and a coulomb times V is equalto a joule. Thus, a joule represents the amount of electrons (amperes)with a driving force (volts) in a closed circuit system per time unit.For calculations of the joule value, the current, potential and timewere all measured in the fuel cells employed.

Example 4 Single Compartment Fuel Cell—Electricity Production By E. Coli

[0067]FIG. 5 compares electricity generation by E. coli in a twocompartment fuel cell as prepared as described above (A, B and C) versusa single compartment fuel cell as prepared as described above (D, E, andF) with different electrode compositions. Potential was higher in thetwo compartment fuel cell; whereas current wasp equivalent in eitherfuel cell system. Current was significantly lower when a woven graphiteanode and a Fe³⁺ graphite cathode were used. Notably, nearly equivalentcurrent levels were obtained when either a neutral red woven graphiteanode or a Mn⁺⁴ graphite anode were used as electrodes in either fuelcell system with a Fe³⁺ graphite cathode.

[0068] Table A summarizes the comparison of electricity production byEscherichia coil with different anode-cathode combinations. E. coilproduced increasing levels of current using Fe⁺³ graphite as the cathodewhen the anode was charged from graphite woven to NR-graphite woven toMn⁺⁴ graphite. The electron transfer efficiency with E. coli wasdramatically higher with Mn⁺⁴ graphite as the anode. When comparingelectrical production E. coli using the neutral red-woven graphiteelectrode developed here versus the system described in Park and Zeikus,“Electricity Generation In Microbial Fuel Cells Using Neutral Red And AnElectronophore”, Appl. Environ. Microbiol., 66:1292-1297, 2000 usingsoluble neutral red and a woven graphite as the anode, there wasobserved a maximum potential of 0.85 volt and a maximum current of 3.9mA/12 g woven graphite (3.14 mA/m² graphite electrode) with solubleneutral red; and, a maximum potential of 0.79 volt maximum current and17.7 mA/2.7 g woven graphite (3.0 mA/m² graphite electrode with neutralred linked woven graphite electrode). The electron transfer of neutralred linked to woven graphite was 10-fold higher than neutral red linkedto a graphite plate reported by Park et al., in “Electricity ProductionIn Biofuel Cell Using Modified Graphite Electrode With Neutral Red”,Biotech. Lett. 22:1301-1304, 2000.

Example 5 Electricity Production By Sewage Sludge

[0069]FIG. 6 compares electricity production by sewage sludge microbesin a two compartment fuel call as prepared above (A, B, C) versus a onecompartment fuel cell as prepared above (D, E, F). Current was higher inthe single compartment fuel cell than the two compartment fuel cell withall anode-cathode combinations tested; whereas, the potential was nearlyequivalent. Sewage sludge bacteria produced significantly higher currentlevels than E. coli for all anode-cathode combinations that were tested(see Table A).

[0070] After the experiments were finished, the anode was removed fromthe one compartment fuel cell and examined it by scanning electronmicroscopy (see FIG. 7). Sample preparation and scanning electronmicroscopy investigation were performed at Michigan State UniversityCenter for Advanced Microscopy, East Lansing, Mich., USA. The graphitefelt was removed at the end of the experiment in the fuel cellcontaining sewage sludge and glucose. The graphite felt electrode sample(cut from electrode surface 5×5×5 mm) was fixed in 4% glutaraldehydebuffered with 0.1 M sodium phosphate at pH 7.4. Following a brief risein the buffer, the sample was dehydrated in ethanol an series (24%, 50%,75%, 95%) for 5 minutes at each gradation and with 3-5 minute changes in100% ethanol. The sample was then critical point dried in a Blazerscritical point dryer, with liquid carbon dioxide as the transitionalfluid. The sample was then mounted on the aluminum stab using epoxy glueand sputter coated with gold in an Emscope Sputter Coater SC500 purgedwith argon gas. The sample was then examined in a JEOL JSM-6400Vscanning electron microscope. The electrode surface was coveredprimarily with a coccoidial morphological type of bacteria. Thediversity of different morphological types observed on the electrodesurface was greatly limited from that observed in the sewage sludgeitself. Apparently specific bacteria attached to the anode and used itas an electron acceptor for growth and energy metabolism. TABLE AComparison of electricity production among four anode-cathodecombinations in biofuel cell systems with resting cells of anaerobicsewage sludge or E. coli as biocatalyst. The mean of electricityproduction Approximate Electron Transfer Biocatalyst Anode CathodeCurrent Potential Efficiency (bacteria) Materials Materials (mA) (V)(mA/m² electrode^(c)) Sewage Woven Graphite^(a) Woven Graphite^(a) 0.340.6 0.268 Sludge Woven Graphite Fe³⁺ Graphite 1.30 0.6 1.024 NR WovenGraphite^(b) Fe³⁺ Graphite 11.0 0.58 8.661 Mn⁴⁺ Graphite Fe³⁺ Graphite14.0 0.45 1750 E. Coli Woven Graphite^(a) Woven Graphite^(a) 0.6 0.60.47 Woven Graphite Fe³⁺ Graphite 1.5 0.35 1.181 NR Woven Graphite^(b)Fe³⁺ Graphite 3.3 0.35 2.598 Mn⁴⁺ Graphite Fe³⁺ Graphite 2.6 0.28 325 #The external shape and size of the three electrodes was the same but theweight and surface areas were different.

Analysis Of Examples 1-5

[0071] Practical improvements have been demonstrated in both microbialfuel cell designs and enhanced microbial electron transfer efficiencieswith new cathode and anode compositions. The new single compartment fuelcell system offers advantages over a conventional two compartment fuelcell. First, the new single compartment fuel cell system is simpler andless expensive to construct and operate, Second, the single compartmentfuel cell system eliminates the need for a ferricyanide catholyte andaeration which might use more energy than the fuel cell makes, Third;the single compartment fuel cell system replaces the expensive protonselective membrane with a porcelain septum. Fourth, the use of a Fe⁺³graphite cathode enhances electron transfer efficiency for a wide rangeof microbes as potential biocatalysts. A current of 14 mV was producedwith sewage sludge using Mn⁴⁺ graphite anode and a Fe³⁺ cathode which isthree times higher than reported using a woven graphite electrode andsoluble neutral red (See, Park et al., “Electricity Production InBiofuel Cell Using Modified Graphite Electrode With Neutral Red”,Biotech. Lett. 22:1301-1304, 2000.).

[0072]FIG. 8 provides a diagrammatic explanation for the differentpotential and current levels obtained when different anode and cathodeconfigurations were used to produce electricity in microbial fuel cellsusing either E. coli or anaerobic sewage sludge. In general, theover-all electron driving force is directly related to the potentialdifference between the anode and cathode. In System 1 where a graphitecarbon anode and cathode are used, it is very difficult to transferelectrons from the bacterial cell to the anode because of the lack of anelectron channel or mediator that interacts with the cell membrane.Consequently, in System 1 current is low but the potential is high. InSystem 2, electrons transfer from the cell to the anode is difficultwithout an electron channel but transfer from the cathode to oxygen isenhanced by Fe³⁺; thus, current produced is higher than in System 1 butthe potential difference is the same. In Systems 3 and 4, electrons areeasily transferred from the cell to the anode because neutral red orMn⁴⁺ serves as an electron channel; but the potential is lower than inSystems 1 or 2.

[0073] Previously, soluble electron mediators were used in fuel cellssuch as neutral red, thionin, and 2-hydroxyl-1,4-naphtoquinone toconvert microbial reducing power into electricity. Most soluble electrondonors except for neutral red cannot be easily bound to cells and allmust be continually added or recycled. This problem can be solved byimmobilizing the electron mediator (e.g. neutral red or Mn⁴⁺) on theelectrode. In the present invention, the immobilization of neutral redto a carbon electrode has been improved by changing from a graphiteplate electrode to a woven graphite electrode which has a surface area1000 times greater. Also in the present invention, it has beendemonstrated that a Mn⁴⁺ graphite plate electrode works as well orbetter than a neutral: red woven graphite in coupling electron transferfrom microbes to electricity production in fuel cells. In sum, these twonew anode compositions significantly enhanced electron transferefficiencies in microbial fuel cells from that reported previously.

[0074] Resting cells in the new fuel cell system described here wereusing lactate as the electron donor and the anode as the electronacceptor for energy metabolism. The Mn⁴⁺ graphite anode proved to be thebest conductor of electron transfer from lactate dehydrogenation incells to the Fe³⁺ graphite cathode. Many different kinds of bacteriaincluding Escheichia Shewanella, Clostridium and Desulfovibrio have beenreported to reduce metallic ions (e.g. manganese, ferric, uranium andcupric) while oxidizing organic substrate (See, Lovley, D. R.,“Dissimilatory Metal Reduction”, Annu. Rev. Microbiol. 47:297-299,1993).

[0075] A variety of microbes have been shown to be electrophilic. It hasbeen reported that metabolizing methanogens can grow with electricity(i.e. the cathode) as the electron donor while reducing CO₂ as theelectron acceptor (see, Park and Zeikus, “Electricity Generation InMicrobial Fuel Cells Using Neutral Red And An Electronophore”, Appl.Environ. Microbiol., 66:1292-1297, 2000). Kim et al. in “DirectElectrode Reaction Of Fe(III)-Reducing Bacterium, ShewanellaPutrefacians”, J. Microbial. Biotechnol., 9:127-13, 1999 demonstratedthat S. putrefacians, which as iron containing cytochromes in its outmembrane, can grow with lactate as the electron donor and a wovengraphite anode as the electron acceptor.

[0076] Here, sewage sludge served as a better biocatalyst than E. colifor electricity generation (See Table A). Without intending to be boundby theory, it is presumed this is because the mixed anaerobic populationcan generate lower reducing power than E. coli because it containsmicrobes whose enzyme co-factors (e.g. F₄₂₀, ferredoxin, etc.) operatebelow −0.4 mv. It can be hypothesized that sewage sludge containselectrophiles (i.e. microbes especially adapted to use an electrode, ashigher electron donors or acceptors for energy metabolism). Theseelectrophilic species may have better electron transfer efficienciesthan E. coli. In this regard, the prevalent coccoidial microbes insewage sludge that dehydrogenated lactate on the neutral red wovengraphite anode did not resemble either E. coli or S. putrefacians.Without intending to be bound by theory, it is believed that the Mn⁴⁺graphite and the neutral red-woven graphite electrodes and fuel cellsdescribed herein may prove useful as “lightning rods” for the enrichmentof electrophiles.

[0077] Thus, the present invention provides a use of Mn⁴⁺-graphite andneutral red-woven graphite electrodes to electrically recycle cofactorsin oxidoreductase linked biocatalysis systems. The electrochemicalbioreactor system using neutral red with resting cells can electricallyrecycle NADH, NADPH and ATP during cellular metabolism and biochemicalsproduction. Furthermore, the electrochemical bioreactor system usingneutral red or Mn⁴⁺ with resting or growing cells can be used to produceelectricity during microbial degradation. Also, the electrochemicalbioreactor system using neutral red or Mn⁴⁺ and oxidoreductases can beused to oxidize or reduce NAD during biochemicals production.

Example 6 Growth of Organisms

[0078]Actinobacillus succinogenes was grown in medium “A” containing 10grams of glucose per liter, 5 grams of yeast extract per liter, 8.5grams of sodium phosphate monobasic per liter, and 10 grams of sodiumbicarbonate per liter under an anaerobic N₂—CO₂ (80:20) atmosphere at37° C. in a 4 liter anaerobic bottle for 16 hours.

Example 7 Preparation of Fumarate Reductase

[0079] Cell extracts were prepared at 4° C. under an anaerobic N₂atmosphere. The harvested and washed cells were resuspended in 100 mMTris—HCl buffer (pH 7.2) containing 1 mM dithiothreitol (DTT) and 0.05mg. of DNase. The bacterial cells were disrupted by passing them twicethrough a French press at 20,000 lb./in.². The cell debris was removedby centrifugation three times at 40,000×g for 30 minutes each time. Thepurified membranes were obtained from cell extracts byultra-centrifugation at 100,000×g for 120 minutes. The clear brownprecipitate was washed twice with 100 mM Tris—HCl buffer (pH 7.2)containing 1 mM DTT and resuspended in the same buffer containing 1 mMDTT by homogenization. The suspended membrane fraction was used as anenzyme source for the fumarate reductase.

Example 8 Electrode Composition and Preparation

[0080] A Fe(III)-graphite electrode was made from mixture of 60% (w/w)fine graphite powder (particle size below 600 mesh), 36% (w/w) kaolin asinorganic binder (particle size below7400 mesh), 3.0% (w/w) ferric ionsand 1.0% (w/w) nickel ions. Distilled water was added to the mixture formaking a graphite paste, and the paste was configured to a square-shapedplate (20 cm.×20 cm.×1 cm. thick) by pressing at 1.0 kg./cm.², drying onair for 48-72 hours at room temperature, and solidifying by baking at1100° C. for 12 hours under anaerobic conditions using a kiln.

Example 9 Preparation of An Electrode Modified with CMC-NR-NAD⁺-FumarateReductase Complex

[0081] A graphite felt electrode modified with CMC-NR-NAD⁺-fumaratereductase complex can be used as an anode or cathode for an electrodereaction according to the invention. A graphite felt electrode modifiedwith carboxymethylcellulose, neutral red, NAD⁺, and fumarate reductasewas prepared as follows. First, a graphite felt electrode was formed asabove in Example 8. The graphite felt electrode was then cleaned bysoaking in methanol for 12 hours and then in deionized distilled waterfor 12 hours. The electrode was dried at 120° C. for 1 hour. Theelectrode was made in the carboxy form by heating at 200° C. for 24-48hours. The electrode was then soaked in dicyclohexylcarbodimide solution(2 mg./ml., in chloroform) at 4° C. for 6 hours, and then dried in air.The electrode was soaked in neutral red (100 μM) solution in chloroformat 4° C. for 12 hours. The electrode was washed by soaking in methanolat 4° C. for 3 hours three times until the unbound neutral red wascompletely removed. The electrode was then dried at 40° C. for 5 hours.

[0082] The electrode was then soaked in a 0.07% carboxymethylcellulose(available from Sigma, C5678, low viscosity, average MW 700,000)solution in deionized distilled water. The electrode was dried at 60° C.for 6 hours until the electrode hardened. The electrode was then soakedin dicyclohexylcarbodimide solution (2 mg./ml., in chloroform) at 4° C.for 6 hours, and then dried in air. The electrode was soaked in neutralred (100 μM) solution in chloroform at 4° C. for 12 hours. The electrodewas washed by soaking in methanol at 4° C. for 3 hours three times untilthe unbound neutral red was completely removed. The electrode was thendried at 40° C. for 5 hours.

[0083] The electrode was then soaked in a 100 mM1-cyclohexyl-3-(2-morpholinoethyl) carboxamide metho-p-toluenesulfonate(CMCD) solution at 4° C. for 6-12 hours. The electrode was then soakedin 1 mM NAD⁺ solution in Tris—HCl buffer (100 mM, pH 7.0) at 4° C. for12 hours. The electrode was then washed in Tris—HCl buffer (100 mM, pH7.0). The electrode was then soaked in a membrane fraction (proteinconcentration 1.635 mg./ml.) isolated from Actinobacillus succinogenesin Tris—HCl (100 mM, pH 7.0) at 4° C. for 12 hours. The treatedelectrode was dried at 4° C in a refrigerator, and then tested for theoxidation-reduction reaction of succinate-fumarate.

[0084] It was believed that 0.35 mg. membrane protein was immobilized on1 gram of graphite felt electrode, which is 0.07% of the total membraneprotein. The concentration of NAD⁺ bound to the graphite felt electrodecan be determined from the concentration of membrane protein bound tographite felt electrode. Because, 1 mM of NAD⁺ was used forimmobilization on the graphite felt electrode, the concentration of NAD⁺bound to electrode was believed to be below 0.0007 mM/G of graphite feltelectrode. The fumarate reductase activity of the membrane protein is9.5 μM NADH/mg. protein/min.

Example 10 Preparation of a Bioelectrochemical Reaction System

[0085] A one compartment electrochemical system suitable for use as abiosensor as shown in FIG. 9 was prepared as follows. An anode wasprepared from 0.05 grams (diameter: 1.0 cm.; thickness: 0.6 cm.; surfacearea: 0.47 m²/g) of the graphite felt electrode modified withcarboxymethylcellulose, neutral red, NAD⁺, and fumarate reductase asprepared in Examples 5-9. A cathode was prepared as a ferric ion (Fe³⁺or Fe(III))-graphite electrode having a surface area of 0.0000785 m²(0.785 cm²). The inside diameter and the height of the reactor were 1cm. and 0.6 cm., respectively. The thickness of the porcelain membraneand the anode were 0.1 mm. and 0.3 mm., respectively. The outside of thereactor was made from a rubber stopper. The bioelectrochemical oxidationof succinate to fumarate was coupled to electricity production, and thereduction of fumarate to succinate was used for biosynthesis. Thecathode surface area used was adjusted to 0.3 g. (1.75×4.0×0.6 cm.,0.47m²g) and the anode surface area was 0.0014m². The volume of each ofthe anode and the cathode compartment was 15 milliliters. The twocompartment system was made from glass, and the porcelain septum(thickness: 3 mm., 3×5 cm.) with micro-pores was used as anion-selective membrane for separation of the anode from the cathodecompartment.

Example 11 Electrochemical Reaction for Detection of Succinate

[0086] Zero, 1, 5, 50, 100, 200, 300 and 400 μM solutions of succinatein Tris—HCl buffer (100 mM, pH 7.0) were prepared for determining lowerdetectable concentration levels of succinate. With the system of FIG. 9,both amperes and potential were measured at the same time after acontinuous flow of 10 ml. of succinate solution from the lowestconcentration to the highest concentration under closed circuitcondition and then without pausing of the current value, the succinatesolution of the next concentration was applied. In addition, 1 to 10 mMof standard succinate solutions in Tris—HCl buffer (100 mM, pH 7.0) wereprepared for determining the catalytic activity of the graphite feltelectrode modified with CMC-NR-NAD⁺-fumarate reductase complex. With thesystem of FIG. 9, the potential was measured after 5 minutes followingthe flow (flow rates, 5 ml. /min.) of succinate solution on an opencircuit system and the current was measured by changing from an opencircuit to a closed circuit system. The highest value of current waschosen and then the current value was paused to zero value bymaintaining of closed circuit system under stopped flow. After pausing,the succinate solution of the next concentration was applied to thesystem. The results are in FIGS. 12 and 13.

Example 12 Oxidation of Succinate to Fumarate Coupled to ElectricityProduction by Electrode Modified with CMC-NR-NAD⁺-Fumarate Reductase

[0087] About 6 mM of fumarate solution in Tris—HCl buffer, (100 mM, pH7.0) was prepared for electricity consumption coupled to reduction offumarate by the modified graphite felt electrode (cathode) with CMC-NRNAD⁺-fumarate reductase that acts as a catalyst. Two control tests weredone. In one control, the modified electrode with CMC-NR-NADH+-fumaratereductase complex was used but no electricity was supplied, and inanother control, 1 mM NADH was added to the reactor but no electricitywas supplied. In the test reaction, the potential between the anode andthe cathode was 2.0 volts and the current was variable from 8 to 10milliamps but NADH was not added to the reactor. The anode and thecathode were separated by the porcelain septum (3 mm. thickness, 3×5)with micropores made from 100% kaolin by baking at 100° C. instead of acation selective membrane (such as that sold under the trademark Nafion,Electrosynthesis). The results are in FIGS. 14 and 15.

Analytical Techniques for Examples 6-12

[0088] In the Examples 6-12, fumarate and succinate were quantitativelyanalyzed by an HPLC (Waters model) equipped with an Aminex Fast Acidcolumn (100 mm.×7.8 mm, available from BioRad, Hercules, Calif., USA)and auto sampler.

Analysis for Examples 6-12

[0089]FIG. 12 shows the linear response rate between a lowerconcentration of succinate detected by the enzyme immobilized biosensorusing the system of FIG. 9. The potential in millivolts and the currentin milliamps were measured on closed circuit without externalresistance. The lowest detection limit of succinate by theCMC-NR-NAD⁺-fumarate reductase modified graphite electrode was 5 μM. Theamount of membrane protein bound to the electrode was 0.35 mg./gram ofgraphite felt. The fumarate reductase activity of the membrane proteinwas 9.5 μM NADH/mg. protein/min.

[0090]FIG. 13 shows the relationship between higher succinateconcentrations and current and potential generated by the immobilizedenzyme chemical sensor. The potentiometric (graph A) and amperometric(graph B) response of the modified electrode with CMC-NR-NAD⁺-fumaratereductase complex was measured at pH 7.0 and at 26° C. using the systemof FIG. 9. Each point of the plot of FIG. 13 corresponds to thepotential value after 5 minutes following the addition of succinate onan open circuit system. The current was measured on a closed circuitsystem. The amount of membrane protein bound to the electrode was 0.35mg./gram of graphite felt. The fumarate reductase activity of themembrane protein was 9.5 μM NADH/mg. protein/min.

[0091]FIG. 14 shows the relationship between electricity generation andthe amount of fumarate produced during succinate oxidation by theimmobilized enzyme electrode. The oxidation of succinate to fumarate onthe graphite felt electrode modified with CMC-NR-NAD⁺-fumarate reductasecomplex, which was coupled to electricity production in an opencircuited (graph A) and closed circuited (graph B) biofuel cell system,was measured. The anode was the graphite felt electrode modified withCMC-NR-NAD⁺-fumarate reductase complex and the cathode wasFe(III)-graphite. The anode surface area was adjusted 0.3 g. (0.47m²/g)and the cathode surface area was 0.0014 m² (14 cm.²). The amount ofmembrane protein bound to the electrode was 0.35 mg./gram of graphitefelt. The fumarate reductase activity of the membrane protein was 9.5 μMNADH/mg. protein/min. Electricity and fumarate production correlatedwith the amount of succinate consumed.

[0092] The reduction of fumarate to succinate on the graphite feltelectrode modified with CMC-NR-NAD⁺-fumarate reductase complex as acatalyst was measured. In graph A of FIG. 15, electricity was notsupplied and NADH was not added to the reactor. In graph B of FIG. 15,electricity was not supplied and 1 mM of NADH was added to the reactor.In graph C of FIG. 15, 2 volts of direct current electrical power weresupplied at 8-10 milliamps. The cathode was the graphite felt electrodemodified with CMC-NR-NAD⁺-fumarate reductase complex and the anode wasFe(III)-graphite. The cathode surface area was adjusted 0.3 g.(0.47m²/g) and the anode surface area was 0.0014 m² (14 cm.²). Theamount of membrane protein bound to the electrode was 0.35 mg./gram ofgraphite felt. The fumarate reductase activity of the membrane proteinwas 9.5 μM NADH/mg. protein/min. FIG. 15 shows that in the absence ofelectricity, the reduction of fumarate to succinate by the immobilizedenzyme electrode was insignificant (graph A), whereas, considerable moresuccinate was produced from electricity (graph C) than from NADH (graphB). This occurred because the electrical reduction of neutral redchemically reduces NAD and both electron carriers are utilized by thefumarate reductase.

Example 13 Growth Of Organisms

[0093]Actinobacillus succinogenes was grown in a medium A (10 g ofglucose per liter, 5 g of yeast extract per liter, 8.5 g of sodiumphosphate monobasic per liter, 10 g of sodium bicarbonate per liter)under an anaerobic N₂—CO₂ (80:20) atmosphere at 37° C. in 4 literanaerobic bottle for 16 hours. Resting cells of Actinobacillussuccinogenes were prepared by harvesting stationary-phase cultures at 4°C. by centrifugation at 5,000×g. The cells were washed twice inresuspended medium (50 mM phosphate buffer, pH 7.0) under anaerobiccondition. Bacterial density was determined by spectrophotometer at 660nm as an optical density (OD). In all experiments bacterial cells wereused right after harvesting without storage in a refrigerator.

Example 14 Preparation Of A Bioelectrochemical System

[0094] A two compartment (anolyte and catholyte) electrochemical cellwas used as a control for comparison of the effects of anode-cathodecombinations on succinate production by bioelectrochemical reduction offumarate. The two compartment electrochemical system was separated by aporcelain septum made from 100% Kaolin, diameter 50 mm and thickness 3mm into an anode and cathode compartment. Fe (III)-graphite (0.0088m²)was used in an anode in all experiments, but either a Mn(IV)-graphite(0.008m²), normal graphite felt (2.8 g, 0.47 m² g) or modified graphitefelt (2.8 g, 0.47 m²/g) with bound NR was used as a cathode forcomparison of the effect on electrochemical reduction of fumarate,respectively. During the experiments, completely anoxic conditions weremaintained in the cathode compartment by gassing with N₂—CO₂ (80:20) gasmixture. The flow rate was adjusted to 300 ml/min. The trace oxygencontained in the mixture gas was removed in a furnace filled with purecopper fillings at 370° C. No gassing and no agitation were used in theanode compartment.

Example 15 Preparation Of Electrodes

[0095] The Fe (III)-graphite anode was made from a mixture of 60% (w/w)fine graphite powder (particle size was below 600 mesh), 36% (w/w)inorganic binder (mainly Kaolin of which particle size was below 400mesh), 3.0% (w/w) ferric ion and 1.0% (w/w) nickel ion. TheMn(IV)-graphite cathode was made from a mixture of 60% (w/w) finegraphite powder, 37% (w/w) inorganiclbinder (Kaolin, white clay), 2.0%(w/w) manganese ion and 1.0% (w/w) nickel ion, respectively. Distilledwater was added to the mixture for making a paste, and the paste wasconfigured into a square-shaped plate (20 cm×20 cm×1 cm thickness) bypressing at 1.0 kg/cm₂, drying on air for 24 hours48 hours at roomtemperature and solidified by baking at 1100° C. for 12 hour underanaerobic conditions using a Kiln.

Example 16 Immobilization Of Neutral Red To Graphite Woven Electrode

[0096] Neutral red was immobilized to the felt electrode by followingprocedure: (1) Cleaning a graphite felt electrode by soaking in methanolfor 12 hours and then in deionized distilled water for 12 hours. (2)Drying the electrode at 120° C. for 1 hour. (3) Making the carboxy formby heating at 200° C. for 24 hours-48 hours. (4) Soaking the electrodein dicyclohexycarbodiimide solution (2mg/ml, in chloroform) at 4° C. for6 hours. (5) Drying the electrode in air. (6) Soaking the electrode inneutral red (100 μM) solution in chloroform at 4° C. for 12 hours. (7)Washing the electrode by soaking in methanol at 4° C. for 3 hours threetimes until the unbound neutral red is completely removed. (8) Dryingelectrode at 40° C. for 5 hours.

Example 17 Electrochemical Reduction Of Fumarate To Succinate By GrowingCells Of Actinobacillus Succinogenes

[0097] A 300 ml sample of fresh medium A with 60 mM fumarate was used asa catholyte and 300 ml of 200 mM KH₂PO4 was used as an anolyte. Afterautoclave, the dissolved oxygen in the catholyte was removed by gassingwith a N₂—CO₂ (80:20) mixture at flow rates of 1 liter/min. for 30 minand then the flow rate was adjusted to 300 ml/min. 30 ml ofprecultivated bacterial cells were inoculated into the catholyte and 2volt electricity was supplied during experiments. Mn(IV)-graphiteelectrode, modified graphite felt with NR and normal graphite feltelectrode were compared to each other. The reactor without an electrodeand NR was used as a control. The soluble (100 μM) was added to thereactor with a normal graphite felt electrode. Two volts of electricitywere used as reducing power. The culture sample was aseptically isolatedfrom reactor and used for analysis after centrifugation at 13000×g for30 min and then by filtration with a membrane filter (pore size, 0.22μM). Fumarate and succinate were quantitatively analyzed by HPLC (Watersmodel) equipped with Aminex Fast Acid column (100 mm×7.8 mm, BioRad,2000 Alfred Nobel drive, Hercules, Calif. (94547)) and an auto sampler.

Example 18 Electrochemical Reduction Of Fumarate To Succinate By RestingCells Of Actinobacillus Succinogenes

[0098] The harvested bacterial cells after 16 hours of cultivation wereused as a biocatalyst. 300 ml of fresh medium A with 60 MM fumarate wasused as a catholyte and 300 ml of 200 mM KH₂PO4 was used as an anolyte.After autoclaving the dissolved oxygen in catholyte was removed bygassing with N₂—CO₂ (80:20) mixture at flow rates of 1 liter/min. for 30min and then the flow rate was adjusted to 300 ml/min. 30 ml ofpre-cultivated bacterial cells were inoculated into the catholyte and 2volt electricity was supplied during the experiments. TheMn(IV)-graphite electrode, the modified graphite felt with NR electrodeand the normal graphite felt electrode were compared to each other. Thereactor without an electrode and NR was used as a control. The solubleNR (100 μM) was added to the reactor with a normal graphite electrode. 2volt electricity was used as a source of reducing power. The culturesamples were aseptically isolated from the reactor and were used foranalysis after centrifugation at 13000×g for 30 min and then filteredwith a membrane filter (pore size, 0.22 μM). Fumarate acid succinatewere quantitatively analyzed by HPLC (Waters model) equipped with AminexFast Acid column (100 mm×7.8 mm, BioRad, 2000 Alfred Nobel drive,Hercules, Calif. (94547)) and an auto sampler.

Example 19 Electrochemical Reduction Of NAD⁺ On Mn(IV)-Graphite Cathode

[0099] 1 mM NAD⁺ was tested for electrochemical reduction coupled tooxidation-reduction reaction of a Mn(IV)-graphite electrode in a 30 mlelectrochemical reactor, 0.0014m². if Mn(IV)-graphite andFe(III)-graphite were used as a cathode and anode, respectively. 0.141m². of graphite felt cathode and 0.0014 m² of Fe⁺³-graphite anode wereused for comparison. Two volts of electricity were applied and thecurrent was varied from 5 to 10 mA.

Analysis For Examples 13-19

[0100] In order for an electron mediator to be effective in electrontransfer from a cathode into microbial metabolism, it needs to be ableto reduce NAD and not be toxic to cells. FIG. 16 shows that the Mn⁺⁴graphite electrode can electrochemically reduce NAD⁺ to NADH. Theelectron transfer efficiency of the Mn⁺⁴ graphite electrode was thencompared to other cathode compositions (Table 1) during fermentation offumarate to succinate by A. succinogenes in a two chamberelectrochemical bioreactor system. Clearly, the Mn⁺⁴ graphite cathodeallowed more electricity to be utilized and more succinate to beproduced than with either the neutral red-bound graphite electrode,soluble neutral red and a graphite electrode, or the graphite electrodealone. When an electron mediator was used, somewhat less cells wereproduced. The Mn⁺⁴ graphite electrode was clearly better at electrontransfer into cells than the other electrodes compositions tested.Therefore, the effect of several physiological parameters on electricityutilization and succinate production by A. succinogenes using the Mn⁺⁴graphite cathode and Fe⁺³ anode were examined. FIG. 17 shows a controlfumarate fermentation by A. succinogenes without electricity and, FIG.18 shows the same conditions but with electricity. It is clear thatelectricity significantly increases succinate production withoutaltering fumarate consumption or growth.

[0101]FIG. 19 shows that the resting cells suspensions (i.e. stationarygrowth phase of O.D. 4.0) can convert fumarate into succinate atsignificant rates.

[0102] Resting cells appeared to electrically reduce fumarate intosuccinate at an optimal 0.9 ratio at 100 mM fumarate and 16 hours (seeFIG. 19) Table 2 compares the influence of different physiologicalparameters on succinate production from fumarate in the absence orpresence of electricity. In the absence of electricity, growing cellsproduce lower levels of succinate than with electricity. Resting cellswithout electricity produce more succinate than growing cells withelectricity and have significantly higher levels of electricityutilization. TABLE 1 Effect Of Electron Mediation System Conditions OnElectrical Utilization, Growth And Succinate Production -(Fe(III)-Graphite Was Used As Anode In All Systems) Electricity FinalSuccinate Productivity Utilized Biomass Concentration Mol succinate/Conditions (mA) OD₆₆₀ (mM) mol fumarate No mediator/ 1.3-1.8 2.46 24.710.556 graphite Soluble NR/ 4.0-4.6 1.91 21.90 0.718 Graphite NeutralRed - 3.3-3.6 1.85 32.71 0.758 Graphite Mn -Graphite 6.3-8.7 1.99 38.600.894

[0103] TABLE 2 Comparison Of Physiological Growth Parameters OnSuccinate Production And Productivity In The Absence Versus The PresenceOf Electricity. Succinate Productivity Electricity Final Succinate (mMbiomass⁻¹, Utilized Biomass Production initial fumarate Conditions (mA)OD₆₆₀ (mM) conc.⁻¹)^(a) Growing cells 0 2.46 24.7 0.1673 withoutelectricity Growing cells 6.3-8.7 1.99 38.6 0.3232 with electricityResting cells 0 3.0 11.5 0.0638 without electricity^(b) Resting cells 8.3-10.6 2.0 42.0 0.3500 with electricity Resting cells  9.5-12.2 4.052.9 0.2204 with electricity

[0104] Therefore, it can be seen that the invention provides anelectrochemical bioreactor system having improved electrodes thatenhance the rate of electron transfer from cells. The electrochemicalbioreactor system has an improved electrode that has utility as anenzymatic fuel cell, as a sensor for succinate detection, and as anengineered catalyst for the synthesis of fumarate or succinate. Theelectrochemical bioreactor system can be used as either an enrichmentdevice for electrophilic microorganisms; that is, those which use anelectrode as an electron acceptor, or electron donor for energyconservation.

[0105] Although the present invention has been described in considerabledetail with reference to certain embodiments, one skilled-in the artwill appreciate that the present invention can be practiced by otherthan the described embodiments, which have been presented for purposesof illustration and not of limitation. Therefore, the scope of theappended claims should not be limited to the description of theembodiments contained herein.

[0106] Industrial Applicability

[0107] The invention relates to improved electrodes for electrochemicalbioreactor systems that can use electrical energy as a source ofreducing power in fermentation or enzymatic reactions and that can useelectron mediators and a biocatalyst, such as cells or enzymes, toproduce electricity.

What is claimed is:
 1. An electrochemical bioreactor system comprising:a first compartment containing a first electrode; a second electrode; anelectrically conductive material connecting the first electrode and thesecond electrode; an electrolyte for providing ionic conductivitybetween the first electrode and the second electrode; and a biocatalystdisposed in the first compartment or associated with the firstelectrode, wherein the first electrode comprises graphite felt and atleast one electron mediator associated with the graphite felt.
 2. Theelectrochemical bioreactor system of claim 1 wherein: the biocatalystcomprises an oxidoreductase and the oxidoreductase is bound to the firstelectrode.
 3. The electrochemical bioreactor system of claim 2 wherein:the oxidoreductase is fumarate reductase.
 4. The electrochemicalbioreactor system of claim 1 wherein: the biocatalyst comprises cells ofActinobacillus succinogenes and the cells are disposed in the firstcompartment.
 5. The electrochemical bioreactor system of claim 1wherein: the biocatalyst comprises cells of Escheichia coli and thecells are disposed in the first compartment.
 6. The electrochemicalbioreactor system of claim 1 wherein: the biocatalyst comprises sewagesludge and the sewage sludge is disposed in the first compartment. 7.The electrochemical bioreactor system of claim 1 wherein: the firstelectrode comprises neutral red covalently bound to the graphite felt.8. The electrochemical bioreactor system of claim 7 wherein: the firstelectrode comprises a carboxylated cellulose bound to the graphite feltand neutral red bound to the carboxylated cellulose.
 9. Theelectrochemical bioreactor system of claim 1 wherein: the firstelectrode comprises NAD⁺ bound to the graphite felt.
 10. Theelectrochemical bioreactor system of claim 9 wherein: the firstelectrode comprises a carboxylated cellulose bound to the graphite feltand NAD⁺ bound to the carboxylated cellulose.
 11. The electrochemicalbioreactor system of claim 1 wherein: the first electrode comprises acarboxylated cellulose bound to the graphite felt, neutral red bound tothe carboxylated cellulose, NAD⁺ bound to the graphite felt, andfumarate reductase bound to the graphite felt.
 12. The electrochemicalbioreactor system of claim 1 wherein: the second electrode includes afirst surface and a second opposed surface, the electrolyte is disposedon the first surface, the first surface faces an interior of the firstcompartment, and the second surface comprises an exterior surface of thefirst compartment.
 13. The electrochemical bioreactor system of claim 1further comprising: an electrical power supply electrically connected tothe electrically conductive material, wherein electrical current isapplied from the electrical power supply to the first electrode and thesecond electrode such that the first electrode acts as a cathode wherebythe electrochemical bioreactor system promotes chemical synthesis in thefirst compartment.
 14. The electrochemical bioreactor system of claim 13wherein: the biocatalyst comprises cells of Actinobacillus succinogenesand the cells are disposed in the first compartment, wherein theelectrochemical bioreactor system promotes the reduction of fumarate tosuccinate in the first compartment.
 15. The electrochemical bioreactorsystem of claim 13 wherein: the first electrode comprises a carboxylatedcellulose bound to the graphite felt, neutral red bound to thecarboxylated cellulose, NAD⁺ bound to the graphite felt, and fumaratereductase bound to the graphite felt, and the electrochemical bioreactorsystem promotes the reduction of fumarate to succinate in the firstcompartment.
 16. The electrochemical bioreactor system of claim 1further comprising: an electrical load electrically connected to theelectrically conductive material, wherein the first electrode acts as aanode and the electrochemical bioreactor system generates electricalcurrent detectable at the load.
 17. The electrochemical bioreactorsystem of claim 16 wherein: the first electrode comprises a carboxylatedcellulose bound to the graphite felt, neutral red bound to thecarboxylated cellulose, NAD⁺ bound to the graphite felt, and fumaratereductase bound to the graphite felt, and the electrochemical bioreactorsystem generates electrical current from the oxidation of succinate tofumarate in the first compartment.
 18. The electrochemical bioreactorsystem of claim 16 wherein: the first electrode comprises neutral redcovalently bound to the graphite felt, and the biocatalyst comprisescells of Actinobacillus succinogenes and the cells are disposed in thefirst compartment.
 19. The electrochemical bioreactor system of claim 16wherein: the first electrode comprises neutral red covalently bound tothe graphite felt, and the biocatalyst comprises cells of Escherichiacoil and the cells are disposed in the first compartment.
 20. Theelectrochemical bioreactor system of claim 16 wherein: the firstelectrode comprises neutral red covalently bound to the graphite felt,and the biocatalyst comprises sewage sludge and the sewage sludge isdisposed in the first compartment.
 21. An electrochemical bioreactorsystem comprising: a first compartment containing a first electrode; asecond electrode; an electrically conductive material connecting thefirst electrode and the second electrode; an electrolyte for providingionic conductivity between the first electrode and the second electrode;and a biocatalyst disposed in the first compartment or associated withthe first electrode, wherein the first electrode comprises a metal ionelectron mediator.
 22. The electrochemical bioreactor system of claim 21wherein: the biocatalyst comprises cells of Actinobacillus succinogenesand the cells are disposed in the first compartment.
 23. Theelectrochemical bioreactor system of claim 21 wherein: the biocatalystcomprises cells of Escherichia coli and the cells are disposed in thefirst compartment.
 24. The electrochemical bioreactor system of claim 21wherein: the biocatalyst comprises sewage sludge and the sewage sludgeis disposed in the first compartment.
 25. The electrochemical bioreactorsystem of claim 21 wherein: the second electrode includes a firstsurface and a second opposed surface, the electrolyte is disposed on thefirst surface, the first surface faces an interior of the firstcompartment, and the second surface comprises an exterior surface of thefirst compartment.
 26. The electrochemical bioreactor system of claim 21wherein: the metal ion electron mediator is an iron cation.
 27. Theelectrochemical bioreactor system of claim 21 wherein: the metal ionelectron mediator is Fe³⁺.
 28. The electrochemical bioreactor system ofclaim 27 further comprising: an electrical power supply electricallyconnected to the electrically conductive material, wherein electricalcurrent is applied from the electrical power supply to the firstelectrode and the second electrode such that the first electrode acts asa cathode whereby the electrochemical bioreactor system promoteschemical synthesis in the first compartment.
 29. The electrochemicalbioreactor system of claim 28 wherein: the biocatalyst comprises cellsof Actinobacillus succinogenes and the cells are disposed in the firstcompartment, wherein the electrochemical bioreactor system promotes thereduction of fumarate to succinate in the first compartment.
 30. Theelectrochemical bioreactor system of claim 27 further comprising: anelectrical load electrically connected to the electrically conductivematerial, wherein the first electrode acts as a anode and theelectrochemical bioreactor system generates electrical currentdetectable at the load.
 31. The electrochemical bioreactor system ofclaim 30 wherein: the biocatalyst comprises cells of Actinobacillussuccinogenes and the cells are disposed in the first compartment. 32.The electrochemical bioreactor system of claim 30 wherein: thebiocatalyst comprises cells of Escherichia coli and the cells aredisposed in the first compartment.
 33. The electrochemical bioreactorsystem of claim 30 wherein: the biocatalyst comprises sewage sludge andthe sewage sludge is disposed in the first compartment.
 34. Theelectrochemical bioreactor system of claim 21 wherein: the metal ionelectron mediator is a manganese cation.
 35. The electrochemicalbioreactor system of claim 21 wherein: the metal ion electron mediatoris Mn⁴⁺.
 36. The electrochemical bioreactor system of claim 35 furthercomprising: an electrical power supply electrically connected to theelectrically conductive material, wherein electrical current is appliedfrom the electrical power supply to the first electrode and the secondelectrode such that the first electrode acts as a cathode whereby theelectrochemical bioreactor system promotes chemical synthesis in thefirst compartment.
 37. The electrochemical bioreactor system of claim 36wherein: the biocatalyst comprises cells of Actinobacillus succinogenesand the cells are disposed in the first compartment, wherein theelectrochemical bioreactor system promotes the reduction of fumarate tosuccinate in the first compartment.
 38. The electrochemical bioreactorsystem of claim 35 further comprising: an electrical load electricallyconnected to the electrically conductive material, wherein the firstelectrode acts as a anode and the electrochemical bioreactor systemgenerates electrical current detectable at the load.
 39. Theelectrochemical bioreactor system of claim 38 wherein: the biocatalystcomprises cells of Actinobacillus succinogenes and the cells aredisposed in the first compartment.
 40. The electrochemical bioreactorsystem of claim 38 wherein: the biocatalyst comprises cells ofEscherichia coli and the cells are disposed in the first compartment.41. The electrochemical bioreactor system of claim 38 wherein: thebiocatalyst comprises sewage sludge and the sewage sludge is disposed inthe first compartment.
 42. A method for electrically recycling cofactorsin an oxidoreductase linked biocatalysis system, the method comprising:using a Mn⁴⁺-graphite electrode in the system.
 43. A method forelectrically recycling cofactors in an oxidoreductase linkedbiocatalysis system, the method comprising: using a neutral red-wovengraphite electrode in the system.
 44. A method for electricallyrecycling NADH, NADPH or ATP during cellular metabolism and biochemicalsproduction in an electrochemical bioreactor system, the methodcomprising: using neutral red with resting cells in the electrochemicalbioreactor system.
 45. A method for producing electricity comprising:using neutral red or Mn⁴⁺ with resting or growing cells in anelectrochemical bioreactor system during microbial degradation.
 46. Amethod for oxidizing or reducing NAD during biochemicals production, themethod comprising: using neutral red or Mn⁴⁺ and oxidoreductases duringbiochemicals production.